The C-terminus of p53 (aa363-393) has been identified as a negative regulatory region for p53 activity. Antibodies against a C-terminal epitope (aa371-380) allow certain p53 mutants to regain DNA-binding activity in vitro and the same effect occurs with deletion of the C-terminus and by other various C-terminal modifications. Thus, it is possible that the wild-type p53 phenotype could be restored to certain mutant forms. Based upon the above concepts we explored a structure-activity relationship in which various C-terminal p53 peptides were correlated to their ability to induce apoptosis in human breast cancer lines. Further testing led to the peptide with greatest ability to induce apoptosis, aa361-382 which was covalently linked to a highly efficient Antennapedia membrane transporter fragment of 17 amino acid residues. Our preliminary results in this proposal showed that this Antennapedia fused p53 derived C-terminal peptide (Ant-p53pep) selectively induced rapid apoptosis in breast cancer cells carrying endogenous p53 mutations or overexpressed wild-type p53, but was not toxic to non-malignant human cells containing wt p53. The peptide-induced apoptosis occurred through a Fas/APO-1 signaling pathway involving increased cell surface levels of Fas/FasL, as well as activation of a Fas/APO-1 specific protease, FLICE. The Ant-p53pep induced apoptosis is p53-dependent since the Ant-p53pep had no effects in three tumor cell lines with null p53. Furthermore, Ant-p53pep bound both mutant and wild type p53 proteins, however, apoptosis was induced only in malignant cells with wild type or mutant p53, but not in non-malignant cell lines. This selective apoptotic effect of Ant-p53pep only in tumor cells, we believe, is dependent upon quantitative differences in p53 protein between malignant (high level of p53) and non-malignant (low level of p53) cells. Thus, the selective and rapid induction of apoptosis occurring only in cancer cells carrying p53 abnormalities could be a powerful therapeutic modality for the treatment of cancer.